Agriculture Reference
In-Depth Information
3.3.3
Initiation of systemic RNA silencing
3.3.3.1 Spontaneous activation of systemic RNA silencing
Systemic silencing was originally discovered in plants exhibiting co-suppression in
a spontaneous manner. In the case of the
Nia
tobacco transformants described above,
however, only a subset of the lines exhibited this property (see Plate 3.1A, B, follow-
ing page 146). These plants were referred to as 'Class-II plants', in contrast to the
'Class-I plants', which were unable to trigger
Nia
silencing
de novo
unless they were
grafted onto silenced scions of Class II, which were competent to send a systemic
silencing signal (Palauqui & Vaucheret, 1998). Molecular analyses indicated that the
difference between Class-I and Class-II lines was not due to transgene copy number
per se
but, rather, to an ill-defined transgene feature that potentiated the capacity
of spontaneous triggering through accumulation of a particular transgene product
(Palauqui & Vaucheret, 1998). Since the construct integrated into the genome of the
Class-II plants was designed to produce sense transcripts, it is very likely that spon-
taneous silencing was elicited via an SDE1-dependent pathway that could sense and
convert aberrant transgene transcripts into dsRNA molecules (see Section 3.3.1.2).
Possibly, the transgene constructs in Class-II plants were more prone to produce
the aberrant RNA than those of Class-I plants. Significantly, transgene dosage was
found to be pivotal in the initiation of silencing in Class-II plants, since analysis
of five independent lines carrying each a single copy of the
Nia
transgene revealed
that only homozygous (as opposed to hemizygous) plants displayed the spontaneous
silencing phenotype (Palauqui & Vaucheret, 1998). Assuming that the sense trans-
gene constructs carried by the Class-II plants were prone to the production of only a
low level of aberrant transcripts, the duplication of the corresponding loci may have
enhanced the chances of such transcripts being detected in the cell and subsequently
processed into dsRNA. Therefore, both qualitative and quantitative features of the
Nia
loci/mRNA influenced the onset of spontaneous silencing.
A detailed analysis revealed that, under standard greenhouse growth conditions,
spontaneous co-suppression was triggered at various frequencies (ranging from 5 to
42%) between individual Class-II lines (Palauqui & Vaucheret, 1995). However, for
all lines, triggering consistently occurred during a phenocritical period ranging from
15 days post-germination to flowering. In addition, the incidence of co-suppression
between individuals from each line was increased if plants were grown
in vitro
prior
to their transfer in greenhouse. Similar observations were made with co-suppressed
lines of tobacco plants expressing a nitrite reductase (
Nii
) transgene (Palauqui &
Vaucheret, 1995). These findings indicate that the physiological state of the plant (i.e.
transition from vegetative to reproductive growth) and environmental factors exert
a critical influence upon activation of spontaneous systemic silencing. In addition,
triggering of
Nia
and
Nii
silencing was found to occur exclusively in mature leaves,
indicating that the intrinsic physiological status of those organs also played an
important role in the initiation of spontaneous silencing (Palauqui & Vaucheret,
1995). In this regard, it is significant that many cells in mature leaves (unlike those
in young developing tissues) undergo the process of endoreduplication, a somatic
